Abstract

It is now scientifically proved that the human immunodeficiency viruses, HIV-1 and HIV-2, are the results of cross-species transmissions of the simian immunodeficiency viruses (SIV) that naturally infect non-human primates in sub- Saharan Africa. SIVsmm from sooty mangabeys (Cercocebus atys atys) is recognised as the progenitor of HIV-2, whereas SIVcpz from chimpanzees (Pan troglodytes troglodytes) and SIVgor from gorillas (Gorilla gorilla gorilla) in West-central Africa are the ancestors of HIV-1, the virus responsible for the AIDS (Acquired ImmunoDeficiency Syndrome) pandemic having already infected more than 60 million people. Only non-human primates (NHPs) species from Africa are infected with SIVs. Serological evidence of SIV positivity has been shown for at least 40 of the 69 primate species found in Africa and this has been confirmed by DNA sequence analysis in 32 species. Generally, SIVs do not induce an AIDS-like syndrome in their natural hosts, suggesting that they have been associated and evolved with their hosts over an extended period of time. However, if SIV crosses the species barrier, it may become pathogenic to the new host. The ancestors of HIV-1 and HIV-2 have crossed the species barrier to humans on multiple occasions, most likely through the contact with infected blood and tissues from primates hunted for bushmeat. Bushmeat hunting has been a longstanding practice throughout sub-Saharan Africa, but the trade has increased in the last decades. Commercial logging has led to the construction of roads into remote forest areas and hunters are now making use of this newly developed infrastructure to penetrate previously inaccessible forests and capture and transport bushmeat to major city markets. Moreover, villages around logging concessions have become more densely populated; this has also increased the trade and consumption around these areas. The socio-economic and environmental changes occurring combined with the growing genetic diversity and SIV prevalence among non-human primate populations, suggest that today, more than previously, the human population is exposed to SIVs. Bushmeat hunting is not limited to chimpanzees, gorillas or sooty mangabeys: the majority of NHPs is represented by many Cercopithecus and Colobus species for example. It is therefore important to continue the search and the characterisation of new SIVs and to determine the prevalence of infection in the NHP, in order to better evaluate which of these SIVs represent a health threat for the human population. The main goal of this thesis were (i) to determine SIV infection and investigate its prevalence among different social groups of monkeys living in Taï National Park, Côte d’Ivoire, knowing that these NHPs are heavily hunted around this area (ii) to determine SIV infection and prevalence and to characterise at a molecular level the SIV possibly infecting the red colobus species found in Abuko Nature Reserve, the Gambia. Two different subspecies of red colobus are found in the Gambia and in Côte d’Ivoire: this represented an opportunity to investigate whether different subspecies may harbour genetically different viruses and therefore to better understand the impact of geographical barriers on the evolution of SIV (iii) to compare the molecular structure of SIVs infecting red and olive colobus, two sister species in the Colobinae subfamily (iv) to determine if the chimpanzee subspecies found in West Africa is infected with a virus similar to that of the monkey species it preys upon, knowing that, to date, only the subspecies from West-central and East Africa have been found to be infected with SIV. The majority of the results presented in this thesis have been obtained by analysing data collected with non-invasive methods. SIV infection has been determined in NHPs by detecting antibodies or by isolating viral sequences from freshly dropped faecal samples collected in the forests of Côte d’Ivoire and The Gambia. In order to be able to discriminate the faecal samples collected and, consequently, to reliably determine the prevalence of infection in a monkey group, the host has been genotyped by analysing the DNA extracted from the epithelial cells debris released from the intestine. Host genotyping, antibody detection and isolation of viral RNA from faecal samples have become possible thanks to the improvement of conservation methods and DNA and RNA extraction techniques. Wild-living non-human primate populations often live in inaccessible areas and tend to be wary of the presence of observers or display cryptic behaviour; the difficulty in sampling increases when the target species are arboreal primates exploiting the higher layers of the forest canopy. To mitigate these problems, we selected two field sites (Abuko Nature Reserve, The Gambia and Taï National Park, Côte d’Ivoire) where the primate populations were at least partly habituated by the presence of human observers and where behavioural-ecology studies have been conducted for more than 10 years. In fact, SIV is transmitted mainly sexually, but possibly also vertically (from the mother to the offspring) and through biting or infection of open wounds. It is therefore important to consider factors such as mating system, patterns of dispersal, group size, average number of adult males in a group, polyspecific associations, etc. as parameters conducive to the transmission of the virus within or across groups and species. In total, more than 300 faecal samples from two groups of western red colobus (Piliocolobus badius badius), from two groups of black-and-white colobus (Colobus polykomos polykomos), from three groups of olive colobus (Procolobus verus), from three groups of Diana monkeys (Cercopithecus diana), from one group of Campbell’s monkeys (Cercopithecus campbelli), from one group of lesser-spot nosed monkeys (Cercopithecus petaurista) and from a group of greater spot-nosed monkeys (Cercopithecus nictitans) were collected near the western border of the Taï Forest, in Côte d’Ivoire, between March and July 2004. To discriminate the faecal samples collected, 16 microsatellite loci were screened in these seven monkey species using cross-specific human markers. Microsatellites are di-tri-tetra-nucleotide tandem repeats, which length’s variability is transmitted by Mendelian inheritance and can therefore be used in combination for individual discrimination. Between 25% to 37% of the primers used were informative and successfully and reliably amplified faecal extracted DNA from all species (Chapter 5). Colobus and Cercopithecus samples were first tested for the presence of HIV cross-reactive antibodies using an immunoblotting assay and were found to be all negative or ‘non interpretable’. Subsequently, Reverse Transcriptase-Polymerase Chain Reactions (RT-PCRs) using universal as well as species-specific primers that target the gag, pol and env regions of the SIV genome were performed: only the western red colobus tested positive for SIV infection. Among the inferred 53 adult individuals belonging to two neighbouring habituated groups, 14 tested SIVwrc (western red colobus) positive with a prevalence of 26%. Phylogenetic analysis of pol and env sequences revealed a low degree of viral genetic diversity in each group. The viral sequences obtained were generally clustering together according to their respective social group of origin. Conversely, the degree of viral genetic diversity between the two groups was higher. Behavioural and demographic data collected previously from these communities indicate that western red colobus monkeys live in promiscuous multi-male societies, where females leave their natal group as sub-adults and where extra-group copulations or male immigration have been rarely observed. Phylogenetic data reflect these behavioural characteristics (Chapter 6). The negative SIV results obtained for the other investigated species may reflect their social structure and mating system, but possibly also the difficulty of group monitoring, faecal sample collection coverage in the field as well as the long term conservation of viral RNA in the field and the sensitivity and specificity of SIV serological and molecular detection tools respectively (Chapter 9). In parallel, sixteen faecal samples from sixteen individuals and two tissue samples from two carcasses of Temminck’s red colobus monkeys (Piliocolobus badius temminckii) collected from the forest floor, between January and February 2005, in the Abuko Nature Reserve were analysed. None of the 16 faecal samples from Temminck’s red colobus analysed by RT-PCR were positive. However, SIV infection was identified in one of the tissue samples, and phylogenetic analyses of partial pol and env sequences showed that this SIVwrc-Pbt virus strain is closely related to SIVwrc-Pbb strains from P.b.badius in the Taï Forest, suggesting that geographically separated subspecies can be infected by closely related viruses. Molecular characterization and phylogenetic analysis of a SIVwrc-Pbt and two SIVwrc-Pbb full-length genomes, subsequently sequenced (Chapter 8), confirmed that SIVwrc-Pbt and SIVwrc-Pbb belong to a species-specific SIV lineage, although distantly related to the SIVlho lineage comprising SIVs from mandrills (Mandrillus sphinx), l’Hoest (Cercopithecus lhoesti) and sun-tailed monkeys (Cercopithecus solatus) (Chapters 7 and 8). More recently, we characterised the full-length genome of the SIV infecting the olive colobus (SIVolc), by analysing a blood sample collected during a previous study also conducted in Taï National Park. Olive and western red colobus are sister taxon and results showed that SIVwrc and SIVolc form distinct lineages, but are closely related across their entire genome (Chapter 8). These results confirmed the complex evolutionary history of primate lentiviruses, which has been driven by host-virus co-speciation, cross-species transmission and recombination events over an extended period of time. Genomic characterization of additional SIVs viruses from other Colobines is needed to better understand the ancestral phylogenetic relationship to SIVs from the l’Hoest lineage and whether recombination occurred between ancestors of these viruses. Finally, 5 blood and tissue samples collected from 5 chimpanzee carcasses during a previous study conducted in Taï National Park, were analysed for SIV infection in this study. To date, no SIV has been isolated from this subspecies of chimpanzee (Pan troglodytes verus) found in West Africa. It is known that the chimpanzee hunts small monkeys and that SIVcpz, the precursor of HIV-1, isolated from the west-central chimpanzee subspecies (Pan troglodytes troglodytes), is a mosaic virus resulting from the recombination of viruses from different species of monkeys the chimpanzee preys upon. Following this line of reasoning, we tested universal as well as species-specific markers amplifying SIV infecting western red colobus, the favourite prey of chimpanzees from the Taï Forest. Serological tests conducted on these 5 samples showed a weak seropositivity in three of them. These results could not be confirmed by PCR. Whether this chimpanzee subspecies is not infected with SIV or whether it harbours a highly divergent virus not detected yet by the current molecular tools available remains to be determined (Chapter 10).